BpWOX11 promotes adventitious root formation in Betula pendula

Adventitious root formation is a key step in vegetative propagation via cuttings. It is crucial for establishing birch plantations and preserve birch varieties. Although previous studies have highlighted role of WOX11 in controlling adventitious root formation, no such study has been conducted in birch. Understanding the mechanism of adventitious root formation is essential for improvement of rooting or survival rate using stem cuttings in birch. In this study, we cloned BpWOX11 and produced BpWOX11 overexpression (OE) transgenic lines using the Agrobacterium-mediated plant transformation. OE lines exhibited early initiated adventitious root formation, leading to increase the rooting rate of stem cuttings plants. RNA sequencing analysis revealed that OE lines induced the gene expression related to expansin and cell division pathway, as well as defense and stress response genes. These may be important factors for the BpWOX11 gene to promote adventitious root formation in birch cuttings. The results of this study will help to further understand the molecular mechanisms controlling the formation of adventitious roots in birch. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04703-z.


Introduction
Vegetative propagation using cuttings is an important method for forest reproduction.The induction of explants to produce adventitious roots (ARs) is a key step for developing full plant [1][2][3].Enhancing success rate relies on promoting the formation and development of ARs in vitro.In vitro plant propagation, external conditions such as temperature, humidity, and hormone levels play crucial roles in influencing AR formation and intermediate branch includes WOX8, WOX9, WOX11, and WOX12 proteins, and WOX11 plays an essential role in AR formation in plants.A loss-of-function mutation in WOX11 leads to a decrease in the number of crown roots (a type of AR) and growth rate in rice (Oryza sativa).The overexpression (OE) of this gene promotes the early occurrence of crown roots and significantly increases root biomass [14].In addition, the presence of the transcription factor WOX11 was required for crown root proliferation induced in OsYUCs(OsYUC3, OsYUC4, OsYUC8, OsYUC10, OsYUC11 and OsYUC14) overexpression [15,16].Inhibition of the WOX11 leads to decrease rooting ability, whereas WOX11 OE increases root formation in Arabidopsis [2,9].WOX11 directly affects damage-induced auxin accumulation in the cambium and its surroundings, upregulates the expression of LBD16 and LBD29, and transforms the leaf cambium or its nearby parenchyma cells into root cells [9].The OE of PeWOX11a or PeWOX11b not only increases the number of ARs on the cuttings but also induces the formation of ectopic roots in the aboveground parts in Populus × euramericana [17].In contrast, LncWOX11a may inhibit the development of ARs by downregulating PeWOX11a [18].Therefore, de novo organ regeneration studies have identified WOX11 as a key gene involved in cell fate transition.WOX11 increases the number of ARs and promotes their formation in isolated plant organs [9,19].
Due to its low cuttings rooting rate, Betula pendula faces challenges in expanding vegetative propagation through cuttings.Although WOX11 has been extensively studied in Arabidopsis, rice, and Populus trichocarpa, functional study of WOX11 in birch is scarce.In this study, we identified the cDNA sequence of BpWOX11 and successfully obtained WOX11 OE transgenic lines.We investigated the rooting index of transgenic lines and performed RNA sequencing (RNA-seq) to identify the differentially expressed genes (DEGs) related to ARs.Our study provides insight into molecular mechanism of ARs and offers the potential application to increase the rooting rate of birch plants through cuttings.

Plant material
These birch (Betula pendula) plants were planted in the Seed Research of Northeast Forestry University (126° 64′ E, 45° 72′ N) in Northeast Forestry University, Harbin, China.The mature fruit was dried; the bracts and pedicels were removed.The seeds were sealed and stored in a refrigerator at − 20 °C.BpWOX11 was cloned from birch and transferred to mature birch seeds.

Cloning and bioinformatics analysis of BpWOX11
The sequence of AtWOX11 was retrieved from the Arabidopsis thaliana database (https://www.arabidopsis.org),and homologous sequence was identified in the birch genome.Based on the mRNA sequence, the upstream and downstream primers of BpWOX11 were designed (Table S1).The cDNA of the wild-type (WT) birch cuttings was used as a template for PCR amplification.The PCR product was purified and ligated using the pCloneEZ vector (pCloneEZ-BpWOX11).After transformation, the monoclonal clones were selected for sequencing at Beijing Qingke Biotechnology Co. Ltd.

Construction of the overexpression vector
The full-length CDS sequence of BpWOX11 was amplified by PCR from pCloneEZ-BpWOX11 and ligated into p35S: BpWOX11 overexpression vector by homologous cloning (2 × 35 S driving BpWOX11, gene terminator is CaMV term, resistance gene is Bar, resistance gene promoter is 35 S, resistance gene terminator is 35 S PolyA) (Catalog.No. VK011-06, Viewsolid Biotech, Beijing, China).Table S1 lists the primer sequences.T4 ligase was used based on the requirements of the vector construction kit.Constructs were incubated overnight at 16 °C.The positive recombinant plasmid (p35S:: BpWOX11) were screened from Glyphosate (5 mg / L) resistance plates.

Genetic transformation of BpWOX11
After sequencing and confirming the correct sequence, the positive recombinant plasmid (p35S:: BpWOX11) was transformed into Agrobacterium EHA105 using the electric shock method to obtain the EHA105 (p35S::BpWOX11) engineered bacteria [22].Transgenic birch plants were obtained via the Agrobacterium-mediated transformation of birch zygotic embryos.Open pollinated seeds were collected from clonal trees (one genotype) in seed orchard of Betula pendula in Northeast Forestry University.A bulb contains a set of seeds, each set of seeds is dried and sealed in a plastic bag and stored in a refrigerator at − 20 °C.The wild type birch (seeds from one bulb for transgenic experiment) seeds were immersed in water for 2 or 3 days, and sterilized in 30% hydrogen peroxide for 15 min and rinsed with sterile water, before seeds were prepared to be used as the transgenic explants.Agrobacterium tumefaciens containing the p35S:BpWOX11 vector was used to infect the longitudinally cut seeds.Eventually, the explants were placed onto co-culture medium (WPM + 0.8 mg/L 6-BA, 0.02 mg/L NAA) under dark for 2 days and then planted on selective medium (WPM + 0.8 mg/L 6-BA, 0.02 mg/L NAA, 5 mg / L glyphosate and 200 mg/L cefotaxime).After 30 days, resistant adventitious buds were induced (WPM with 0.8 mg/L 6-BA, 0.02 mg/L NAA, 0.5 mg/L GA3,5 mg / L glyphosate and 200 mg/L cefotaxime) and rooted (WPM + 0.4 mg/L IBA), half of the uninfected seeds were cultured in initial culture medium (WPM + 0.8 mg/L 6-BA, 0.02 mg/L NAA) for 32 days and finally in induction medium (WPM + 0.8 mg/L 6-BA, 0.02 mg/L NAA and 0.5 mg/L GA3) for 32 days to obtain the WT line [22].The composition of the medium is listed in Table S2.The temperature of the tissue culture room was maintained at 25 °C ± 2 °C, a photoperiod of 16/8 h and 1000-1500 lx.The DNA from transgenic and WT birch lines was extracted by using extraction kit (DNAquick Plant System, TIANGEN, Beijing, China).Transgenic lines were identified through PCR and primers are shown in Table S1.The primer sequence were designed using specific sequences based on p35S:BpWOX11, which include one end of the gene and part of the vector [22].

Quantitative reverse transcriptase-PCR (qRT-PCR) of transgenic birch lines
Total RNA was extracted from leaf of transgenic plants and WT lines using the RNeasy Plant Mini Kit (Biotake) and reverse transcribed into cDNA using the ReverT-reAce® qPCR RT Kit (Toyobo, Osaka, Japan).The SYBR Green PCR master mix (Toyobo) was used for qRT-PCR in the ABI 7500 Real-Time PCR system.Bp18S rRNA and BpActin was used as the internal reference gene, and gene-specific primers were designed using the Oligo7 software (Table S3).The results were analysed using the 2 −ΔΔCT method.Each reaction was repeated three times [22].

AR growth of transgenic plants under tissue conditions
Three transgenic lines (OE3, OE6, and OE8) were selected for rooting experiments.Eighteen plants with similar growth statuses were selected from each line for rooting culture in rooting experiment.Six plants were placed into each bottle containing rooting medium.To investigate the rooting ability of the transgenic BpWOX11 plants, the rooting medium was deprived of indole-3-butyric acid.The rooting rate was measured at 14 and 28 days.The number and length of the ARs of the three OE and WT lines were determined at 28 days.

Greenhouse evaluation of root cuttings experiment
One-year-old plants of OE3, OE6, OE8, and WT were planted for the cuttings experiment in the greenhouse.Cuttings without apical buds, each measuring 6-8 cm in length and having one leaf at the top, were utilized.The experiment divided into three replications, with 30 cuttings used in each replication.Automatic sprinkler irrigation system was used, maintaining the culture environment at a daytime temperature of 28 °C ± 2 °C and nighttime temperature of 22 °C ± 2 °C with 70-80% humidity.At day 30, rooting rate, as well as the number and length of the ARs were determined.

RNA-seq and differential gene expression analysis
RNA-seq samples were collected from the bases of 0-and 15-day cuttings from OE6 and WT from greenhouse.Each treatment comprised three biological replicates and each replicate contained 30 cuttings.RNA was extracted by CTAB method from 12 samples [23].The quality of RNA was detected by 1% agarose gel, NanoDrop 2000, and Agilent 2100 Bioanalyzer.Oligo (DT)-magnetic beads were used to enrich poly (a) mRNA.Fragment buffer was used to fragment the poly (a) mRNA.The fragments were reverse transcribed into cDNA library using test kit (Illumina, San Diego, United States).Read mapping, quality control, and functional annotation were obtained from the BGISEQ-500 platform.Low-quality reads, defined as these with more than half of the component bases in the reads having a quality score of < 15, were filtered out using SOAPnuk (v1.4.0; parametric-l: 15 -q 0.2 -n 0.1) software.Other low-quality regions such as adapter sequences and regions with an excessive number of unknown bases (annotated as "Ns") were also filtered by SOAPnuk.The remaining clean reads were mapped onto the birch reference genome (https://genomevolution.org/coge/api/v1/genomes/35080/sequence) using the Hierarchical Indexing for Spliced Alignment of Transcripts HISAT (v2.1.0)system [24].Bowtie2 was used to align clean reads to the reference coding genomic sequence and the fragments per kilobase of transcript per million mapped reads (FPKM) was calculated by RSEM (v1.2.12) [25].Differentially expressed genes (DEGs) were identified by DEseq2 (v1.4.5) with the criteria of | log2 Fold Change| ≥ 1, and a false discovery rate (FDR) <0.05 [26].Gene annotation used NCBI/Nr (http://www.ncbi.nlm.nih.gov),NCBI NT (http://www.ncbi.nlm.nih.gov), Swiss-Prot (http://www.uniprot.org/keywords/),and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg).The target genes were annotated in the Gene Ontology (GO) database (http://www.geneontology.org/) and then classified into their corresponding functional categories for study of biological functions.

BpWOX11 overexpression lines were obtained by genetic transformation
In the birch genome, Bpev01.c0441.g0004.m0001was identified as BpWOX11, a homologous gene of AtWOX11 in Arabidopsis thaliana, with a similarity of 49.31%.The CDS of BpWOX11 was 744 bp, encoding a protein of 247 amino acids, belonging to the homeodomain superfamily family (Fig. 1A).BpWOX11 CDS amplification showed that the amplified sequence was of 750 bp (Fig S1).After the purification, ligation, and transformation of E. coli, a single clone was selected for sequencing.The results showed that the amplified sequence was 744 bp, consistent with the results obtained using the NCBI database.Further protein sequence alignment and phylogenetic tree analysis of BpWOX11 were performed (Fig. 1B and  C).BpWOX11 showed high similarity to WOX11 across several species, with the highest homology, 64.50% with GhWOX11 of cotton.
BpWOX11 OE lines (OE1-OE8) were obtained by Agrobacterium-mediated zygotic embryo transformation (Fig. 2A and B).DNA was extracted from leaves of ten-month-old transgenic BpWOX11 overexpression lines (OE1-8) and control (WT).The positive plasmid and eight transgenic lines amplified a single band near 750 bp, which was consistent with BpWOX11 sequence adding a section of vector sequence length (840 bp).The WT DNA did not show an amplified band (Fig. 2C).The WT and eight (OE1-OE8) positive lines are shown in Fig. 2D.
RNA was extracted from leaves of ten-month-old transgenic BpWOX11 overexpression lines (OE1-8) and control (WT).Gene expression of BpWOX11 in OE1-OE8 lines was significantly higher than that in WT lines (p < 0.05).OE6, OE3, and OE8 lines was 84.14, 40.69, and 27.86 times higher than that in WT lines, respectively (Fig. 2E).The three lines were selected for subsequent experiments.

BpWOX11 improves AR formation and rooting rate for propagation by cuttings
Measurements of AR growth under tissue culture conditions showed that the rooting rate of the OE lines was significantly higher than that of the WT lines without exogenous hormone application at day 14 (Fig. 3A).No significant difference was observed in the rooting rate and AR number between the OE and WT lines at day 28 (Fig. 3A and B).However, AR length of OE lines was significantly higher than that of the WT lines (Fig. 3c).At day 30, growth rates relative to pre-rooting (0d) were significantly higher for the OE2, OE5, and OE7 lines than for the WT line, whereas no significant differences were observed for the other transgenic lines (Fig S3).
In the greenhouse cuttings experiment (Fig. 3D), results show the rooting rate was 33.33% for WT, whereas OE3, OE6, and OE8 lines were 61%, 72% and 67%, respectively (Fig. 3E).The number and length of AR for OE6 and OE3 lines were significantly higher than that in the WT lines.The root lengths for OE3 and OE8 lines were significantly lower than that of the WT lines (Fig. 3F and G).

RNA-seq analysis
The expression BpWOX11 gene increased leading to elevated rooting rate of OE lines in birch cuttings.The bases of cuttings from OE6 and WT lines with the highest rooting rate were used.RNA-seq analysis was performed at two timepoints, day 0 and day 15, and three biological replicates were established (Fig. 4A).We identified 8770 and 8338 differentially expressed genes (DEGs) before and after cuttings in the WT and OE lines, respectively.These DEGs were called AR formation genes.Among them, 3662 were upregulated and 5048 were downregulated in the WT lines, whereas 3849 were upregulated and 4489 were downregulated in the OE lines.A large proportion of the upregulated (57.93%) and downregulated (63.33%) genes was shared between the WT and OE lines (Fig. 4B and C).The proportion of AR-related DEGs shared between the OE and WT lines increased by 79.65% and 81.59%, respectively, before cuttings (day 0) and during cuttings (day 15) (Fig. 4D).In the OE lines, up-regulated (393/1337) and down-regulated (318/1159) did not change significantly at day 15 (Fig. 4E).Results indicated that gene expression induced AR formation was conserved in the OE and WT lines.
Cuttings also induced a specific set of up-regulated (811, 65.88%) and down-regulated (364, 51.05%) genes between WT and OE lines (Fig. 4E).At day 15 vs. day 0, upregulated genes were 1067 and 887 for OE lines and WT lines, whereas downregulated genes were 775 and 1327 for OE lines and WT lines, respectively (Fig. 4B).The average gene expression for OE lines was higher than that in the WT lines (p < 0.05) (Fig. 4F, Table S4).These data indicated that high WOX11 expression strongly responded to changes in gene expression during AR formation.The GO enrichment analysis showed that 811 upregulated genes and 364 downregulated genes were identified for the OE lines after rooting (Fig. 4G, Table S5  and S6).The upregulated genes were mainly enriched in cell division, cell wall biosynthesis, developmental processes, and cell morphology.The cell division pathway included genes that promoted AR formation and development, namely SCRSCARECROW (SCR) and SHORT ROOT (SHR) (Fig. 4H).WOX11 upregulated the expression of a group of expansin genes in the plant cell wall loosening pathway (Fig. 4H).In addition, GO enrichment showed that a specific set of up-regulated genes (811 genes) between OE and WT were also enriched in the response to stress pathway after rooting.The genes enriched in this pathway mainly consisted of five gene families related to stress response, i.e., UDP-glycosyltransferase, WRKY transcription factor, LEAF RUST 10 DISEASE-RESISTANCE LOCUS RECEPTOR-LIKE PROTEIN KINASE, G-type lectin S-receptor-like ser-ine_threonine-protein kinase, Cytochrome P450(Fig.4I).qRT-PCR showed that rooting-related transcription factors (SCR and SHR) in the cell division pathway were consistent with the RNA-seq results (Fig S4).The activation of cell division-related genes and expansins after WOX11 OE may increase the occurrence of ARs during birch cuttings, thereby improving the rooting efficiency of the cuttings.

BpWOX11 plays a role in the early rooting stage
Rooting rate, root length, and root number have been used as indicators to evaluate vegetative propagation [28][29][30][31].In this study, WOX11 overexpression led to improve the rooting rate of birch cuttings, as results of the early initiation of ARs and increase number of Ars.In Arabidopsis, WOX11 OE has been reported to significantly increase the number of adventitious roots and decrease the length of main roots [9,14].WOX11/12 plays a major role in the first event of rooting cell establishment.The expression of WOX11/12 was decreased in the second step of cell fate transition, whereas those of WOX5 and WOX5/7 increased [32].Therefore, we speculate that BpWOX11 plays a major role in the early stage of de novo organogenesis in birch.Our results showed that overexpression BpWOX11 accelerated the formation of new ARs in cuttings and increased the number of ARs; however, it did not promote the continuous growth process after AR formation.

BpWOX11 promotes rooting as result of regulating genes related to cell wall tissue and cell division
Expansin, as a cell wall relaxation protein, plays a crucial role in the root development of soybean (Glycine max) and Arabidopsis [33,34].In Arabidopsis, expansin genes (AtEXP7 and AtEXP18) are closely associated with the initiation of root hairs.In plants, root hairs originate from epidermal cells which are accompanied by local cell wall loosening [35].In this study, BpWOX11 increased rooting rate of birch.RNA-seq analysis indicated that WOX11 induced a group of expansins in the plant-type cell wall loosening pathway.Therefore, BpWOX11 may promote the growth of ARs by inducing the upregulation of expansin to promote cell wall loosening.
WOX11 promoted the expression of genes associated with cell division, such as KIN7B, TSK, SHR, and SCR [36][37][38][39].SHR and SCR are crucial for regulating the process of asymmetric cell division in root tissue [40].SCARECROW-LIKE gene increases its mRNA levels during the first 24 h induction of root from stem cuttings in Pinus radiata D. Don and Castanea sativa Mill [41].The expression pattern of PrSHR in Pinus radiata is similar to that of the SCARECROW-LIKE gene, and it promoted the formation and maintenance of root meristem and the cambium area of hypocotyl cuttings [42].These results indicated that SHR and SCR play a crucial role during the earliest stage of AR formation.Therefore, we considered that WOX11 promotes AR formation in the early stage of birch cuttings by promoting the expression of SHR and SCR.

Overexpression of BpWOX11 promotes the expression of stress-related genes
In Arabidopsis, 18% of the DEGs in the AtWOX11 mutant plants were involved in stress response compared with those in the WT plants.[43].In this study, transcriptome analysis of OE6 and WT strains revealed that overexpression of WOX11 directly upregulated the expression of stress-related genes.WOX11 can control root hair development and also play a role in increasing the drought resistance of rice [16].Therefore, WOX11 may also be involved in regulating the expression of stress-related genes in birch.We found that 393 genes were upregulated, and 318 genes were downregulated in BpWOX11 OE lines at day 0 and 15 (Fig. 4E).The upregulated 393 DEGs were enriched in defense response, response to stress, and stimulation (Table S7).The downregulated 318 differential genes were enriched in phosphorylation, cell recognition, phosphorus metabolism, and defense response(Table S8).Moreover, at day 15, WOX11 alone upregulated 207 genes involved in response to stimulus and 129 genes involved in response to stress in OE lines (Table S5).Our results showed that OE lines could be used for improving the drought resistance in the early stage of cuttings without roots in birch.

Fig. 1 Fig. 3 Fig. 2 Fig. 4 (
Fig. 1 Sequence and phylogenetic analysis of BpWOX11.(A) BlastX analysis of the BpWOX11 sequence; (B) BpWOX11 amino acid sequence alignment with other that of WOX11 other species; (C) BpWOX11 and WOX11 amino acid sequence clustering analysis of different species

(Fig. 4
See figure on previous page.)Transcriptome sequencing of overexpression (OE) and wild-type (WT) cuttings.(A) Schematic diagram of the cuttings (Created By BioRender.com);(B) The number of upregulated and downregulated of differentially expressed genes (DEGs) for the OE and WT lines in the cuttings process; (C) Overlapping DEGs between WT and OE lines in the cuttings process; (D) DEGs related to rooting-related genes for OE and WT lines.The Y-axis shows the percentage of rooting related genes before and after cuttings; (E) Upregulated or downregulated rooting-related genes before and after cuttings for OE lines; (F) The log 2 expression ratio of the co-activation or co-inhibition genes for OE and WT lines at day 15/day 0; (G) Interval-based GO enrichment analysis; (H) Heatmap of DEGs in cell division and plant cell wall loosening pathways; I Heatmap of DEGs in response to stress pathways